Current views on HIV-1 latency, persistence, and cure

Zora Melkova1,2 & Prakash Shankaran1& Michaela Madlenakova1,2 & Josef Bodor2

Received: 10 May 2016 /Accepted: 20 September 2016# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2016

Abstract HIV-1 infection cannot be cured as it persists inlatently infected cells that are targeted neither by the immunesystem nor by available therapeutic approaches.Consequently, a lifelong therapy suppressing only the activelyreplicating virus is necessary. The latent reservoir has beendefined and characterized in various experimental modelsand in human patients, allowing research and developmentof approaches targeting individual steps critical for HIV-1 la-tency establishment, maintenance, and reactivation. However,additional mechanisms and processes driving the remaininglow-level HIV-1 replication in the presence of the suppressivetherapy still remain to be identified and targeted. Current ap-proaches toward HIV-1 cure involve namely attempts to reac-tivate and purge HIV latently infected cells (so-called Bshockand kill^ strategy), as well as approaches involving gene ther-apy and/or gene editing and stem cell transplantation aiming atgeneration of cells resistant to HIV-1. This review summarizescurrent views and concepts underlying different approachesaiming at functional or sterilizing cure of HIV-1 infection.

Introduction

HIV/AIDS infection can be successfully treated and con-trolled with the combined antiretroviral therapy (cART)

affecting different steps of HIV-1 replication cycle. Thus,HIV-1 infection could be viewed as a chronic disease with arelatively long life expectancy. However, cART, which is stillnot available to all in need, cannot cure HIV infection due tothe presence of latently infected reservoir cells. The latentproviral DNA cannot be recognized by the immune systemnor targeted by cART. Consequently, a lifelong therapy isnecessary, which is expensive and leads to various complica-tions and treatment failures. Therefore, new approaches to-ward functional or sterilizing cure are intensively explored –namely attempts to reactivate and purge HIV-1 latently infect-ed cells (so-called Bshock and kill^ strategy), as well as ap-proaches involving gene therapy and/or gene editing and stemcell transplantation aiming at generation of cells resistant toHIV-1. The ongoing research focuses especially on the mech-anisms of establishment and maintenance of the latent reser-voir, assessment of its size and composition, as well as onstimulation of the innate and specific immunity to promoteHIV-1 clearance.

HIV-1 latency

Retrovirus replication cycle is specific by a step of reversetranscription and a consequent stable integration of the provi-ral DNA into the host cell genome. Depending on the status ofthe host cell, either the HIV-1 provirus can be immediatelyexpressed and the virus replication cycle can proceed furtheror the provirus can become dormant and wait until the latentlyinfected cell encounters the right stimulus (often a specific,possibly rare antigen) and becomes activated. It is thevery presence of the latently infected cells that makes HIV-1infection incurable as these cells serve as a source of virusrebound after a discontinuation or failure of the antiretroviraltherapy. After the activation of the host cell or specific

* Zora Melkovazmelk@LF1.cuni.cz

1 Department of Immunology and Microbiology, 1st Faculty ofMedicine, Charles University, Studnickova 7, 128 00 Prague2, Czech Republic

2 BIOCEV, Biotechnology and Biomedicine Center of the Academy ofSciences and Charles University in Vestec, Průmyslová 595, 25250 Vestec, Czech Republic

Folia MicrobiolDOI 10.1007/s12223-016-0474-7

changes in the epigenetic regulation, the chromatin status andavailability of transcription and other factors change and HIV-1 replication can restart (Fig. 1).

HIV-1 persistence during therapy

HIV-1 infects namely CD4+ T lymphocytes but also myeloidcells like macrophages, microglia, astrocytes, and dendriticcells, even though to a lesser extent. Acute infection ofCD4+ T cells usually leads to cell death, but occasionally,these cells survive and revert back to a resting memory state.Alternatively, latency may be established directly in restingCD4+ T cells. Resting memory cells, including the latentlyinfected ones, then persist and/or are replenished by homeo-static proliferation. In contrast, macrophages are resistant tocytopathic effects of HIV-1 infection and support virus persis-tence in various anatomical sanctuaries as tissue resident mac-rophages (Kim and Siliciano 2016; Kumar et al. 2015;McKinstry et al. 2010).

After initiation of cART, plasma viremia and the level ofHIV-1-infected cells in peripheral blood decay with a wellcharacterized kinetics based on populations with a differentturnover contributing to plasma viremia (Hilldorfer et al.2012) (Fig. 2). A rapid decline of plasma viremia duringfirst two phases indicates the efficiency of antiretroviral

drugs. Phase I represents the turnover of the free virus(half-life from minutes to hours) and mainly of productivelyinfected CD4+ T cells (half-life of 1–2 days; Ho et al. 1995;Perelson et al. 1997; Perelson et al. 1996; Wei et al. 1995).Consequently, phase II corresponds to the decay of cellsmore resistant to HIV-induced cytopathic effect (partiallyactivated T cells and cells of the monocyte-macrophage lin-eage with half-life of 2–4 weeks; Perelson et al. 1997; Shanand Siliciano 2013). The following phase III with a very lowdecay kinetics (half-life of 273 days) proceeds after severalyears into phase IV with a remaining stable low level plasmaviremia that does not decrease any more (Hilldorfer et al.2012; Maldarelli et al. 2007). Despite the very low plasmaviremia below the limit of clinical assays (<50 copies/ml),which is detectable with only ultrasensitive assays (about 3copies/ml), cell-associated proviral DNA (prDNA) and RNA(caRNA) are commonly detected by PCR-based assays inperipheral blood mononuclear cells (PBMCs) during thisphase (Palmer et al. 2008; Palmer et al. 2003) (Fig. 2).The presence of the phase IV indicates the existence of ad-ditional cell populations that do not succumb to virus-induced cytopathic effects and/or either are refractory tocART (proviral DNA or transcription cannot be targeted bycART) or persist in anatomical sanctuaries that are notaccessed by cART. Reactivation of the latently infected cells,which both persist and are replenished by homeostatic

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Fig. 1 Latency and reactivation(simplified scheme). HIV-1latency is characterized by arepressed chromatin, presence ofhistone deacetylases (HDACs),histone methyltransferases(HMTs), and DNA methylases(DMTs), as well as lack oftranscription factors, resulting in atranscription block. Reactivationis associated with epigeneticchanges that lead to openchromatin structure, namelypresence of histoneacetyltransferases (HATs),nuclear translocation oftranscription initiation factors NF-κB and NFAT, increased levels ofTat and formation of its complexwith p-TEFb. Tat-p-TEFbcomplex binds to TAR RNA,resolving promoter-proximalpausing of RNAP II and allowingefficient transcription elongation.p-TEFb can be sequestered in the7SK snRNP inhibitory complex

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proliferation (Chomont et al. 2009; Kim and Siliciano 2016),is viewed as the most relevant source of the new HIV RNA(Shan and Siliciano 2013). However, lower levels ofantiretrovirals (ARV) found in anatomical/immunologicalsanctuaries like brain, genital tract, gut mucosa, or lymphnodes can support the ongoing HIV replication (Fletcheret al. 2014; Huang et al. 2016; Massanella et al. 2016).Based on sequence determination of HIV-1 present in lymphnodes and blood at several time-points after initiation ofcART, the mathematical modeling of virus evolution andtrafficking between the two compartments with low and highlevels of ARV, respectively, supported the view that evolu-tion of drug-sensitive viruses in lymph nodes can keepreplenishing the viral reservoir despite the apparently effi-cient cART (Lorenzo-Redondo et al. 2016). Lately, it wasdescribed that SIV/HIV-1 may continue to replicate within Bcell follicles due to follicular exclusion of CD8+ T cells anddysregulated responses of follicular regulatory T cells (TFR)and follicular T helper cells (TFH; Fukazawa et al. 2015;Miles et al. 2015; Saison et al. 2014; Tran et al. 2008).Furthermore, in addition to latently infected long-lived mem-ory T cells and tissue resident macrophages (Avalos et al.2016; Gludish et al. 2015), an evidence for existence oftissue resident memory T cells (TRM) emerged (Farber2015; Farber et al. 2014).

Another cause of the residual low-level HIV replicationunder cART could involve the immune hyperactivation in-duced by various mechanisms. In addition to the residual pro-ductive infection that is likely to rise immune responses, thereis a more frequent (95 %) abortive infection of resting non-permissive CD4+ T cells. Due to the cytoplasmic DNA-sensing mechanisms like IFI-16, cGAS, and PQBP-1 (Gaoet al. 2013; Thompson et al. 2014; Yoh et al. 2015), these cellsmight die by pyroptosis, a very inflammatory type of pro-grammed cell death (Doitsh et al. 2014; Monroe et al. 2014).Furthermore, HIV-1 replication and disruption of the intestinal

barrier lead to microbial translocation, stimulation of innateimmune responses, and depletion of CD4 Th17 cells, a defectpersisting even after initiation of cART (Chege et al. 2011;Schuetz et al. 2014). Finally, the activation and dysregulatedfunction of regulatory T cells (Tregs), critical modulators ofimmune responses, apparently also contribute the immunehyperactivation (Mendez-Lagares et al. 2014; Saison et al.2014). The residual low-level HIV replication that leads toan elevated immune activation thus further stimulates HIVreplication, generating a vicious cycle.

In summary, HIV persistence during cART is supported byhomeostatic and antigen-induced proliferation of latently in-fected cells, ongoing replication in the sanctuaries as well asby increased immune activation and inflammation (Chomontet al. 2009; Fukazawa et al. 2015; Kim and Siliciano 2016;Van Lint et al. 2013). Additionally, the latent reservoir is read-ily being replenished during episodes of viremia due to atreatment failure or other conditions of incomplete or missingpharmaceutical control.

Models of HIV-1 latency

Our knowledge of the mechanisms, maintenance, and reacti-vation of the latent reservoir is based namely on variousin vitro and in vivo models as the presence of latently infectedcells in human body is very low, around 1–10 millions in thewhole organism, and difficult to study (Crooks et al. 2015;Finzi et al. 1999; Massanella and Richman 2016; Silicianoet al. 2003).

Individual models of HIV latency reproduce to some extentcertain aspects of the complex situation in vivo and allow forstudies of certain latently infected populations or specific as-pects of latently infected cells. Models used to study HIV-1latency in vitro include HIV-infected cell lines (immortalizedlymphocytic or monocytic cells like J-Lat, ACH-2, U1;

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Fig. 2 Comparison of HIV-1RNA and DNA decay curvesupon introduction of cART. Afterinitiation of cART, plasmaviremia and the level of HIV-1infected cells in peripheral blooddecay with a well characterizedkinetics based on populationswith different turnoverscontributing to plasma viremia(Phase I–IV). HIV-1 DNA revealsa relatively smaller decrease.Adapted from Hilldorfer et al.(2012) and Margolis et al. (2016)

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Clouse et al. 1989; Folks et al. 1989; Folks et al. 1987; Jordanet al. 2003), primary cell models (derived from HIV-1 nega-tive donor PBMCs by infection with a particular HIV-1 isolateor a recombinant), and resting CD4 cells (derived fromHIV-1-infected patients; Spina et al. 2013). While the cell lines arecommonly available and easy to handle, they reveal severalaspects that often make them behave differently from the sit-uation in vivo (namely, clonality based on the integration siteor mutations of specific HIV-1 or host cell sequences).

Primary cell models might seem to be closer to the situationin vivo, while the percentage of latently infected cells avail-able for further studies is much higher than in vivo. Differentprimary cell models developed by different laboratories useeither resting or activated CD4+ T cells that are infected withwild-type (typically NL4-3) or recombinant HIV-1 (often ex-pressing a fluorescent reporter gene like EGFP) and treatedwith combinations of several cytokines or chemokines. Afterestablishment of the infection, latency is induced by ARVsfollowed by reactivation by different means. The individualmodels differ in the target cell population, percentage of la-tently infected cells generated, type and time of readout(Bosque and Planelles 2009; Gondois-Rey et al. 2006;Lassen et al. 2012; Saleh et al. 2007; Spina et al. 2013;Tyagi et al. 2010; Yang et al. 2009b). Nevertheless, it is ques-tionable how closely these models reflect the situation in vivo,and how much they are biased due to the experimental setupand mode of readout.

Resting CD4 cells derived from HIV-1-infected patientscultured and stimulated ex vivo thus seem to be the mostrelevant in vitro model. However, their use is limited by a verylow presence of latently infected cells, high background rateof defective integrated proviruses, and difficulties of any anal-ysis due to a very high background of uninfected cells (prob-lems with sensitivity and specificity).

The in vivo models of HIV latency include namely differ-ent types of humanized mice and macaques infected with HIV,SIV, or various recombinants. The advantage of humanizedmice consists in their relative affordability and ease of han-dling; on the other hand, their preparation is tedious whilegraft-versus-host disease (GvHD) and other differences,namely due to their genetic background, the way of immunereconstitution with human tissues, and lineage precursors,may limit their use and relevance of the results.

Humanized mice used for HIV research were originallybased on SCID mice (severe combined immunodeficiencymice) that were irradiated and then transplanted with fetalhuman thymus and liver (SCID-hu Thy/Liv). In this model,latent infection is established during thymopoiesis (deactiva-tion phase), leading to generation of latently infected naïveCD4+ T cells. Among other limitations, this model does notprovide an efficient peripheral reconstitution and human cellsare found in relatively small numbers (Brooks et al. 2001;Marsden et al. 2012).

Lately, BLT mice (bone marrow-liver-thymus humanizedmice) are considered as a better model for complex studies ofHIV reservoirs and latency reversing agents (LRA) as theyprovide a robust peripheral reconstitution. These mice aremost commonly based on irradiated NSG mice (NOD/SCID-gamma chain null mice) transplanted with fetal humanthymus and liver and then reconstituted with bone marrow orpurified CD34+ stem cells (Donahue and Wainberg 2013).This particular combination results in high-level systemic re-constitution of all human leukocyte lineages with improved Tcell maturation and selection in a thymic environment and ingeneration of latently infected naïve and resting CD4+ T cells(Denton et al. 2012). There are also modifications of thesemice using reconstitution with only discrete cell types like Tcells or macrophages (T cell-only mice (ToM); myeloid-onlymice (MoM); Honeycutt et al. 2013; Honeycutt et al. 2016),allowing studies of the role of the individual cell types in theestablishment of latent reservoir or reactivation.

Although the use of BLT mouse model is valuable for theHIV studies, its important limitation consists in the develop-ment of GvHD, typically around 6 months after engraftment(Karpel et al. 2015). Further, these mice are unable to developproper HIV-specific adaptive immune responses consisting inhigh levels of hyper-mutated, class-switched IgG antibodiesas human cells of non-hematopoietic origin, namely thosegiving rise to stromal cells, are not transplanted, and second-ary lymphoid organs do not properly develop (Malhotra et al.2013; Wang et al. 2011).

Yet another BLT model, based on human Rag2−/−γc −/−

mice (Choudhary et al. 2012; Traggiai et al. 2004; Zhanget al. 2007), was recently developed and led to identificationof central memory CD4+ T cells (TCM) as the main latentlyinfected population after suppressive cART similarly as inhuman (Choudhary et al. 2012; Donahue and Wainberg2013). This model was further improved by generation ofhumanized TKO-BLT mice (triple knockout-BLT mice) on aC57Bl/6 background (C57BL/6 Rag2−/−γc−/−CD47−/−) inwhich the GvDH was much reduced, complement was func-tional, and secondary lymphoid organs with a well organizedarchitecture and virus-specific immune responses were devel-oped (Lavender et al. 2013).

A model closest to HIV-1 infection in human is infection ofnon-human primates (NHP), namely rhesus macaques, withvarious SIV strains. It is believed that different SIVs crossedthe species barrier into humans many times, namely SIVsmmnaturally infecting sooty mangabeys and SIVcpz infectingchimpanzees resulted in HIV-2 and certain clades of HIV-1(Hirsch et al. 1989; Huet et al. 1990; Sharp and Hahn 2011).

The NHP models reveal many important features compa-rable to HIV-1-infected humans like anatomy, physiology, im-mune system, infectious agent itself, and susceptibility to an-tiretroviral treatment (Gardner and Luciw 2008; Policicchioet al. 2016). The use of NHP models has been essential for

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understanding pathogenesis of HIV/AIDS as well as for stud-ies of different therapeutic and vaccination approaches. Manyconclusions could be also inferred by comparing immune re-sponses to SIV infection in the natural and unnatural hosts,sooty mangabeys and rhesus macaques, respectively, in whichthe SIV infection results in non-pathogenic or pathogenic con-ditions. There is a high viremia in both hosts but a very littleloss of CD4+ T cells and preservation of architecture andfunction of lymph nodes in the natural host. In the gut of thenatural host, CD4+ T cells are only moderately depleted andnumbers and functions of Th17 cells remain preserved, whilemicrobial translocation is lacking. Further, TFH and TCM areonly weakly infected (Ploquin et al. 2016).

Additionally, recombinant viruses like RT-SHIV or SHIVcontaining different parts of HIV-1 genome can be used toovercome certain differences between HIV-1 and SIVs, e.g.,in studies involving inhibitors specific for HIV reverse tran-scriptase (Jiang et al. 2009; Ndung’u et al. 2001). One impor-tant difference in comparison with HIV-1 is that most SIVs(and HIV-2) encode a vpx protein that allows virus infectionalso in non-dividing cells as it targets for degradation cellularSAMHD-1, an enzyme that would hydrolyze dNTPs and thusdecrease reverse transcription (Hofmann et al. 2012). Despitemany advantages, a major drawback of using macaques con-sists in their cost and ethical and legal regulations of their use.

Obviously, the most relevant approach to study HIV laten-cy and reactivation is in HIV-infected human patients in clin-ical studies. However, performance of clinical studies is strict-ly regulated, expensive, and must be preceded by extensivepre-clinical testing (including animal models). Additionally,human studies never provide really homogeneous and repro-ducible experimental settings with all necessary controls.

In summary, the results obtained in various in vitro andin vivo models and in human patients indicate that the latentHIV-1 reservoir is represented mainly by latently infectedresting CD4+ Tcells, long-lived central memory CD4+ Tcells(TCM cells), and transitional memory CD4+ T cells (TTM

cells). In addition to these cells, latent reservoir may comprisealso other cell populations like CD34+ hematopoietic progen-itor cells, naïve CD4+ T cells, CD4+ memory stem cells(TSCM cells) or γδ T cells, as well as myeloid cells likemacrophages and dendritic cells (Buzon et al. 2014; Carteret al. 2010; Chomont et al. 2009; Honeycutt et al. 2016;Soriano-Sarabia et al. 2015; Wightman et al. 2010). The im-portance of these cells constituting the latent reservoir consistsin their different survivals and stabilities as well as in differentrequirements for signaling and activation and thus HIV-1 pro-virus induction (Archin et al. 2014b).

Assessment of the size of the latent reservoir

Under suppressive cART, plasma viremia (virion-associatedunspliced RNA) is undetectable or below the detection limit ofcommon commercial assays (<50 copies/ml). However,markers of immune hyperactivation persist and cell-associated HIV RNA can be readily detected both in periph-eral blood and tissues like lymph nodes, tonsils, gut, or testes.Thus, in order to determine the effect of treatment intensifica-tion strategies and namely efficiency of LRA explored fortherapeutic reactivation, availability of specific and sensitivemethods allowing an accurate assessment of the size of thelatent reservoir is crucial (Fig. 3).

The latently infected cells can be defined as cells harboringquiescent, replication-competent provirus. A gold standard indetermination of latently infected cells is a quantitative virusoutgrowth assay (qVOA) that is very material-, labor-, andcost-demanding. It requires large volumes of patients’ bloodto isolate sufficient numbers of highly purified latently infect-ed resting CD4+ T cells and plate them in serial dilutionsalong with activated donor PBMCs (CD8 negative) or witha MOLT4/CCR5 cell line. It requires a 2–3-week incubationwith changes of medium and other additives (Finzi et al. 1997;Massanella and Richman 2016; Siliciano and Siliciano 2005).

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Fig. 3 PCR-based detection ofHIV-1 RNA and DNA.Determination of cell-associatedmultiply spliced RNA (msRNA)and proviral DNA (prDNA) usingtwo-step seminested (RT-) qPCRor using a combination of alimiting dilution assay and one-step nested RT-PCR (e.g.,TILDA; Bullen et al. 2014;Kiselinova et al. 2014; Procopioet al. 2015)

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However, this assay apparently underestimates the size of thelatent pool, as it has been already demonstrated that consecu-tive rounds of stimulation/reactivation of the isolated cellscould lead to increased proportion of the reactivated provi-ruses (Bruner et al. 2015; Ho et al. 2013). Further, only 1–2% of the body’s lymphocytes are present in peripheral blood,while most of them are found in tissues and the distribution oflatently infected cells might not be proportional (Blum andPabst 2007; Massanella and Richman 2016). The real size ofthe latent reservoir thus might be at least 60× higher thanobserved in a common qVOA, complicating the effort to cureHIV using latency reversal (Bruner et al. 2015; Ho et al.2013). Importantly, about 10 % of HIV-1 proviruses that re-main silent after the maximal stimulation were found to befully replication competent, suggesting that they might bereactivated in vivo upon different conditions than those usedex vivo during qVOA. Also, the induction of intact proviruseswas proposed to be stochastic, dependent on the levels of Tat,but independent of the cellular activation status (Ho et al.2013; Weinberger et al. 2005; Weinberger et al. 2008).

An analog of qVOA, mouse virus outgrowth assay(MVOA), allows to determine virus outgrowth in humanizedmice. It has been recently described to detect latently infectedcells with higher sensitivity than the standard qVOA as a largenumber of cells can be used and a GvHD promotes HIV-1reactivation. Nevertheless, it provides only qualitative results(Metcalf Pate et al. 2015).

At the other side is a PCR-based determination of the cell-associated DNA, which apparently overestimates the size ofthe latent pool, as most of the integrated proviruses are mutat-ed or incapable of reactivation for unknown reasons (Ho et al.2013; Sanchez et al. 1997). Depending on the primer/probesequences, total, integrated, or 2-long terminal repeat (2-LTR)circular DNA can be commonly detected (Murray et al. 2014;Pasternak et al. 2013). Finally, determination of cell-associated RNA (caRNA) using various combinations ofPCR-based techniques like seminested RT-qPCR or ddPCR(Bullen et al. 2014; Kiselinova et al. 2014) seems to provide amore relevant marker of viral persistence and/or reactivation.However, it is important to use approaches distinguishing be-tween commonly present prematurely terminated short gagtranscripts, multiply spliced transcripts (msRNAs), andunspliced RNA (usRNA). Levels of usRNA are generallyhigher and therefore better detectable than msRNA. On theother hand, determination of msRNA (e.g., tat/rev; Pasternaket al. 2008) or correctly terminated viral transcripts (usingprimers/probe detecting the polyadenylated tail; Shan et al.2013) better correlates with the ability of the cell to produceinfectious viruses (Bullen et al. 2014; Pasternak et al. 2013).

Based on the PCR detection of tat/rev spliced transcripts, anew quantitative assay allowing single-cell based determina-tion of the inducible viral reservoir called TILDA (Tat/rev-induced limiting dilution assay) was recently presented. The

advantage of this assay is use of only 10 ml of blood, seriallimiting dilutions allowing detection of even single positivecell, and a good correlation with qVOA (Procopio et al. 2015).

A disadvantage of all PCR-based assays is their inability todifferentiate between RNA that might remain retained in thenucleus and for this or other reasons not to be translated into aprotein and RNA giving rise to HIV proteins that could bepresented on the cell surface or constitute new virions (Lassenet al. 2006). Therefore, determination of cell-free RNA(cfRNA) or p24 Ag in culture supernatant better estimatesvirion production. On the other hand, proportion of releasedvirions and detectable cfRNA is much lower in comparisonwith caRNA (1.5 and 7 %, respectively; Cillo et al. 2014).

This problem can be partially solved also by a new assaytermed Prime Flow RNA that combines cell-based detectionof proteins with antibodies and detection of intracellular RNAwith specific probes. It was reported to detect one infected cellin 104–105 cells (comparable numbers are found in peripheralblood of patients on cART) (Romerio and Zapata 2015).

Apparently, highly sensitive assays for determination of HIVproteins in culture supernatant are necessary for a better assess-ment of the efficiency of LRA. One such fully automated assaybased on a Quanterix Simoa technology could detect low levelsof p24 Ag in cell lysates (3 pg/ml; Howell et al. 2015). Anotherapproach might employ TaqMan chemistry-based protein as-says (https://www.thermofisher.com/cz/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/real-time-pcr-protein-analysis/protein-expression-taqman-assays.html#workflow).

In summary, assays based on virus outgrowth are very la-borious and generally underestimate the size of the latent poolas their efficiency can be increased by repeated cycles of in-duction of reactivation. On the other hand, technically easierPCR-based detection of HIV-1 prDNA, or caRNA, overesti-mates the size of the latent reservoir, as most of viral DNA ismutated or not transcribed for unknown reasons, while not allRNA transcripts yield functional viral proteins and/or virions.Detection of virion RNA, cfRNA, or viral proteins in culturesupernatant is more accurate but less sensitive due to lowerlevels of these products. Highly sensitive assays for determi-nation of viral proteins are therefore needed.

Mechanisms of establishment and maintenanceof the latent reservoir

There are two types of HIV latency. A pre-integration latencythat occurs after infection of non-permissive cells, which isshort-lived as unintegrated viral DNA is recognized by cyto-plasmic DNA sensors like cGAS or IFI-16 leading to activa-tion of interferon and inflammasome responses (Gao et al.2013; Thompson et al. 2014; Yoh et al. 2015). On the otherhand, the post-integration latency occurring in cells that

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become quiescent after the infection is the cause of virus per-sistence. The latent reservoir is established very early duringthe HIV-1 infection and its size can be limited by an earlyintroduction of cART. It consists of both T cells and myeloidcells (Marban et al. 2007; Wires et al. 2012), and it is verystable with half-life determined by qVOA of about 44 months(Crooks et al. 2015; Siliciano et al. 2003).

A key determinant of the future fate of the HIV-1 provirusis the site of its integration. Most commonly, sites of HIV-1insertion are found in intragenic regions of actively tran-scribed genes (Lewinski et al. 2006; Serrao and Engelman2016). Perhaps for this reason, the majority of repressed butinducible proviruses are also located within the introns of theexpressed genes (Lewinski et al. 2005; Shan et al. 2011). Theestablishment of HIV latency is thus regulated independentlyof the control of expression of the target host genes (Mbonyeand Karn 2014).

Insertion of the HIV-1 provirus in the actively transcribedgenes may result in transcriptional interference, contributing tothe regulation of HIV-1 latency. Divergent orientation of thecellular promoter and viral LTR can lead to the lack of recruit-ment of transcription factors, while convergent promoters maylead to a collision of the transcription machinery and prematuretermination of HIV-1 transcription. Parallel orientation of theHIV LTR located downstream of the cellular promoter can leadto the viral promoter occlusion by a read-through transcriptionfrom the cellular gene, displacing key transcription factors onthe HIV LTR (Han et al. 2008; Lenasi et al. 2011). In latentlyinfected cells, a preference for a parallel orientation of the pro-moters was observed, while there was no preference in acutelyor persistently infected cells, suggesting that transcriptional in-terference may be one of the important factors in the establish-ment and maintenance of HIV-1 latency (Shan et al. 2011).

HIV-1 itself does not encode any specific transcription re-pressors, but high levels of HIV-1 transcription activator Tatand its interactions with a cellular cofactor p-TEFb, resolvingpromoter-proximal pausing of RNA polymerase II (RNAP II)are absolutely critical for the provirus expression (Fig. 1;Yamada et al. 2006). In the absence of Tat-pTEFb complex,transcription efficiency decreases drastically and only shortGag transcripts are generated (Kao et al. 1987; Lassen et al.2004; Price 2000). Transcriptional silencing of the provirusesthus results from a series of epigenetic and non-epigeneticchanges occurring at the promoter region and from processesduring the transcription initiation and elongation phases thatdecrease levels of Tat and availability and/or binding of cel-lular factors (Mbonye and Karn 2014).

In the activated CD4+ T cells, which are productively in-fected most often, the intracellular milieu with high levels ofcellular transcription factors, namely transcription initiationfactors NF-κB, nuclear factor of activated T cells (NFAT),and AP-1, drives HIV expression (Mbonye and Karn 2014).However, as the host cell returns to the resting memory

phenotype, cytoplasmic sequestration of these factors causesa significant decrease of transcription initiation at the HIVLTR and allows transcriptional silencing of the provirus, pos-sibly with help of transcriptional inhibitors (Bodor 2006; Heand Margolis 2002; Tyagi and Karn 2007).

The chromatin structure of the proviral 5′ LTR is a criticalparameter in control of HIVexpression. Regardless of the siteof insertion, 5′ HIV LTR is occupied by nucleosomes Nuc-0and Nuc-1 in specific positions at the start site, imposing ablock on RNAP II initiation (Verdin et al. 1993). Several neg-ative DNA-binding factors (e.g., CBF1, YY1, LSF, BRD2,p50 homodimer; He and Margolis 2002; Karn 2013; Tyagiand Karn 2007; Williams et al. 2006) then facilitate recruitingof other repressor complexes and histone- and DNA-modifying enzymes at both core promoter and enhancer re-gions. Histones of the nucleosomes at the 5′ LTR of silentproviruses are deacetylated and trimethylated, which are fea-tures of the repressive heterochromatin. Specifically, atrimethyl mark on histone H3 lysine 27 (H3K27me3), gener-ated through the action of the polycomb repressive complexPRC2, is a mark of the facultative heterochromatin responsi-ble for reversible silencing of various inducible genes andcontributes to viral quiescence, including HIV-1. On the otherhand, H3K9me2/3 is linked with the formation of the consti-tutive heterochromatin during development as well as with theestablishment of HIV-1 latency (Friedman et al. 2011;Maricato et al. 2015; Matsuda et al. 2015). Further, histonemethyltransferases (HMTs) can be found associated with thelatent proviral LTR (du Chene et al. 2007; Friedman et al.2011; Imai et al. 2010; Keedy et al. 2009; Lusic et al. 2013).Namely, a dominant HMT EZH2 constitutes part of the PRC2complex which serves as a binding platform for additionalchromatin-modifying enzymes, histone deacetylases(HDACs), and DNA methyltransferases (DNMTs)(Chenget al. 2011; Friedman et al. 2011; Tae et al. 2011; Vire et al.2006). Methylation of DNA (CpG islands; Bednarik et al.1990) at transcription start site has been suggested to be themost stable modification of the latent provirus LTR. It mightstabilize DNA and prevent provirus reactivation (Blazkovaet al. 2009; Kauder et al. 2009). Lately, relative frequency ofproviruses with a higher LTR DNA methylation was sug-gested to be increased by a prolonged ARV treatment or mul-tiple rounds of reactivation (Trejbalova et al. 2016).

An additional block in HIVexpression may consist in post-transcriptional processes inhibiting HIV-1 protein expression.Namely, both unspliced and spliced HIV-1 transcripts may beretained in nuclei. The export of HIV-1 transcripts is supportedby binding of Rev to Rev response element (RRE) present inpartially spliced and unspliced genomic HIV-1 RNAs and bythe interactions with exportin 1 (Crm-1), a nuclear export fac-tor. Further association with polypyrimidine tract-binding pro-tein (PTB) and related factors seems to affect the export effi-ciency. Thus, unavailability of either factor may promote the

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retention of HIV-1 RNAs in the nucleus (Kula et al. 2013;Lassen et al. 2004; Lassen et al. 2006; Zolotukhin et al. 2003).

Further, several cellular miRNAs have been reported tomodulate HIV-1 expression by targeting essential cellular co-factors involved in HIV-1 transcription like PCAF and cyclinT1. The former is targeted bymiR175p andmiR-20awhile thelatter by miR27b and miR198 (Sung and Rice 2009; Tribouletet al. 2007). There are also several cellular miRNAs (e.g.,miR-28, miR-125b, miR-150, miR-223, and miR-382) thatrecognize the 3′-end of HIV-1 mRNAs, repressing their ex-pression in resting CD4+ T cells (Huang et al. 2007). Finally,there are miRNAs derived from viral sequences (vmiRNAs),namely from TAR and Nef (miRTAR5p/3p and miRN367,respectively; Bennasser et al. 2004; Klase et al. 2007;Omoto et al. 2004; Schopman et al. 2012). Both cellular andviral miRNAs may cause HIV-1 RNA degradation or ineffi-cient expression of HIV-1 proteins.

Mechanisms of reactivation

The reactivation of the functional, inducible latent provirusdepends on the chromatin status and availability of the cellulartranscription factors (Fig. 1). Upon appropriate stimulationand nuclear translocation, NF-κB and NFAT can bind toHIV LTR if the chromatin landscape of the promoter regionis favorable (Bhatt and Ghosh 2014; Lusic et al. 2013; Ott andVerdin 2013). The accessibility of the HIV LTR can be affect-ed by methylation of CpG islands (Bednarik et al. 1990) orbinding of other transcription factors like Sp1 that are able topromote the chromatin configuration favorable for binding ofthe main transcription factors. In fact, Sp1 is required for theformation of the pre-initiation complex and interacts withNF-κB (Perkins et al. 1993). NF-κB and NFAT probably bindin a mutually exclusive, possibly sequential way, as their HIV-1 LTR-binding sites overlap (Giffin et al. 2003; Mbonye andKarn 2014). NF-κB is found in cell lines and primary naïve Tcells, while NFAT is typically present in memory T cells(Dienz et al. 2007). In primary memory T cells, NFAT andLck are required for optimal latent virus reactivation andHIV-1 can be activated in an NF-kB-independent way bytranscription factor DVII-Ets-1, without causing significantT cell activation (Bosque et al. 2011; Bosque and Planelles2009; Yang et al. 2009a). On the other hand, NFAT is dispens-able in Jurkat cell models. NF-κB is commonly activated byPMA or TNF-α, while NFAT is activated by calcium/calcineurin signaling (Bosque and Planelles 2009; Chanet al. 2013; Kim et al. 2011).

Both NF-κB and NFAT recruit CBP/p300 and other histoneacetyltransferases (HATs) (Garcia-Rodriguez and Rao 1998) tofurther acetylate Nuc-1 and to attract SWI/SNF chromatin re-modeling complex. After the minimal initiation andtranscription through the TAR element, RNAP II pauses. If

Tat and p-TEFb, composed of CDK9 and cyclin T1(Herrmann and Rice 1995; Wei et al. 1998) are available tobind and form a complex with TAR RNA hairpin and the tran-scription machinery, kinase activity of p-TEFb mediates phos-phorylation of negative elongation factors (DSIF and NELF)and of RNAP II and allows formation of a superelongationcomplex and continuation of transcription further into the elon-gation phase (Fig. 1). Thus, Tat transactivation and its interac-tion with p-TEFb is absolutely necessary for the efficienttranscription elongation of the HIV provirus. When notcomplexed with Tat, pTEFb is sequestered and held inactivein the transcriptionally inactive 7SK RNP complex containing7SK small nuclear RNA (7SK snRNA), inhibitory factorHEXIM1 and RNA binding proteins LARP7 and MePCE.Yet, Tat may be out-competed by BRD4 in binding top-TEFb, leading to p-TEFb targeting to transcription of cellulargenes (Mbonye and Karn 2014).

Besides this conventional view of events underlying theHIV-1 reactivation, it has been suggested that the decisionbetween HIV-1 replication and latency can be determined bya stochastic noise in gene expression similarly as in other cell-fate decisions. In this view, the probability of HIV-1 reactiva-tion is increased by a higher rate and variation of fluctuationsin HIV-1 gene expression and further stimulated by Tat-mediated positive feedback mechanism (Dar et al. 2014; Hoet al. 2013; Weinberger 2015; Weinberger et al. 2005;Weinberger et al. 2008).

Latency reversal and purging HIV-1

Latently infected cells represent the major barrier to HIV-1cure (Donahue and Wainberg 2013). The major reservoir isconsidered to reside in resting memory T cells, and therefore,this population is in the focus of most efforts to decrease thesize of the latent reservoir (Bruner et al. 2015).

The initial attempts to eradicate HIV-1 latently infectedcells were first described by Fauci et al. (Chun et al. 1998;Chun and Fauci 1999). Later, the term shock and kill strategywas introduced (Archin et al. 2012; Deeks 2012). In short, itconsists in the attempts to reactivate a dormant provirus silent-ly present in latently infected cells, namely long-livedmemoryCD4 Tcells, that would lead to death of the cells harboring thelatent provirus and decrease the size of the latent pool in thepresence of cART. Originally, it was assumed that virus reac-tivation could be achieved with a single agent, namely HDACinhibitors (HDACi) or PKC inducers, and that the replicationof the reactivated virus and the virus-induced cytopathic ef-fects would be sufficient to kill the host cells and thus decreasethe size of the latent reservoir. Today, it is largely accepted thatcombinations of two or more agents with different mechanismof action together with an additional stimulation of anti-HIVimmune responses would be necessary. Namely, improvement

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of cytotoxic responses using engineered dual-affinity (DART)and broadly specific antibodies (bNAbs) toward HIV-1 areintensively investigated (Caskey et al. 2015; Sloan et al.2015). Still, there are doubts if the HIV cure can be everachieved because of unknown fraction of the cells remain-ing refractory to the reactivation strategies. Further, it hasbeen suggested that a prolonged antiretroviral treatmenttogether with random or intentional cycles of reactivationmight lead to an increased methylation of HIV-LTR andthus stabilization of the latent provirus (Trejbalova et al.2016). Therefore, another logical approach would be toinhibit the spontaneous reactivation of latent HIV-1(Mousseau et al. 2015).

Activity of LRA is usually identified in vitro in tissue culturescreens involving various cell lines and primary cells. The effi-ciency of the selected compounds must then be verified in vivo.Most intensively investigated LRA, namely those in clinicaltrials, involve compounds in use or under development fortreatment of various cancers and other diseases. These are es-pecially HDACi and PKC agonists (NF-κB inducers). Thereare 11 HDACs subdivided in four classes (Mottamal et al.2015; Wang et al. 2009). Particularly, HDACs 1–3 (class I)seem to be important in maintaining HIV latency (Keedyet al. 2009). HDACi non-specifically activate transcription ofmany genes by increasing acetylation of the promoter regions,including HIV LTR. Increased acetylation should modify chro-matin status and allow for binding of transcription factors,namely NF-κB and NFAT. Among these, vorinostat (SAHA)has been the most extensively explored, but newer compoundswith higher potency like givinostat, panobinostat, orromidepsin seem more promising (Banga et al. 2016). Theability of individual HDACi alone to induce HIV-1 reactivationex vivo, in cells isolated fromHIV-1-infected patients on cART,apparently depends on specific experimental conditions as wellas on patients’ history, as not all attempts ex vivo were success-ful (Blazkova et al. 2012; Bullen et al. 2014; Rasmussen et al.2013). However, lately, vorinostat, panobinostat, romidepsin,and namely givinostat were found effective in a modifiedVOA employing a prolonged or repeated treatment with indi-vidual HDACi (Banga et al. 2016), repeating the results ofclinical studies, in which vorinostat and some other HDACiwere able to increase levels of caRNA (Archin et al. 2014a;Rasmussen et al. 2014; Sogaard et al. 2015). Nevertheless inthese studies, the size of the latent reservoir was not founddecreased in vivo, suggesting that combination with otheragents or strategies increasing immunological killing of theinfected cells would be necessary (Rasmussen et al. 2013).Other classes of chromatin-modifying enzyme inhibitors in-clude inhibitors of histone methyltransferases (HMTi) likechaetocin, BIX-01294 or GSK343, or inhibitors of DNAmeth-yltransferases (DNMTi) like 5-aza-2′deoxycytidine. However,they are more likely to be effective in combination with otherLRA (Kumar et al. 2015).

The other important classes of LRA represent PKC ago-nists that induce activation and nuclear translocation NF-κBand p-TEFb. They can also trigger activation of MAPK andnuclear translocation of AP1. The natural PKC inducer effec-tive in HIV-1 reactivation is TNF-α, a cytokine found in-creased in untreated HIV-1 infection. However, due to itspro-inflammatory pleiotropic effects, its use as a LRAin vivo cannot be considered. Similarly, the other very effec-tive agent of this class, phorbol myristate acetate (PMA), can-not be considered for therapy as it reveals a strong tumorigenicpotential (Kumar et al. 2015). However, newer PKC inducersinclude non-tumorigenic phorbol ester prostratin (Biancottoet al. 2004; Kulkosky et al. 2001), macrolide lactonebryostatin-1 (Mehla et al. 2010), or diterpenoids ingenol Band ingenol-3-angelate (Jiang et al. 2014; Jiang et al. 2015).PKC inducers downregulate expression of cell surface recep-tors CD4, CXCR4, or CCR5 in uninfected cells, thus limitingthe spread of the newly released virus (Hezareh et al. 2004;Jiang et al. 2014; Mehla et al. 2010). Further, PKC was report-ed to phosphorylate HEXIM1, suggesting that PKC inducersmight affect also this inhibitory protein (Fujinaga et al. 2012).Hexim phosphorylation is commonly mediated by AKT ki-nase that can be stimulated by HMBA (Contreras et al. 2007),leading to the release of p-TEFb from the inhibitory complex7SK RNP. Yet another compound affecting availability of p-TEFb is JQ1, bromodomain inhibitor affecting factors BRD2and BRD4 (Boehm et al. 2013).

Disulfiram, the inhibitor of acetaldehyde dehydrogenaseused in therapy of alcohol abuse, induces degradation ofPTEN, again allowing AKT-mediated phosphorylation ofHEXIM1 (Doyon et al. 2013; Xing et al. 2011). This compoundhas been described as an effective LRA in vitro in a relativelyartificial model of primary CD4+ T cells immortalized with theBcl-2 protooncogene. However, the expectations of its potencywere not fulfilled as there was no significant effect on the size ofthe latent reservoir found in vivo (Spivak et al. 2014).

As mentioned above, combinations of several LRA arelikely to act more efficiently and cause fewer negative sideeffects. The examples of such combinations are chromatin-remodeling compounds, namely HDACi, together with differ-ent inducers of transcription. Chromatin remodeling com-pounds can be apparently considered as noise enhancers thatare relatively ineffective in HIV-1 reactivation alone, whilethey reveal a synergismwith real transcriptional activators thatare able to increase HIV-1 expression alone (e.g., PKC in-ducers) (Dar et al. 2014; Kumar et al. 2015).

Apart from the mainstream studies, there are many otherapproaches toward latency reversal and HIV cure. Of these,use of a pro-oxidant gold-based drug Auranofin is of interestas it was shown to induce a partially selective killing of TCMand TTM CD4+ T cells, the main reservoir cells containingthe latent HIV-1 (Lewis et al. 2011). Consequently, it wasdemonstrated that TCM and TTM CD4+ T lymphocytes are

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more susceptible to the redox stress and apoptosis (Chirulloet al. 2013).

Our laboratory has demonstrated independently thatNormosang, a hemin containing compound used to treat acutehepatic porphyria, could strongly potentiate reactivation of thelatent provirus induced by PKC inducers like PMA, TNF-α,prostratin or bryostatin-1, while it inhibits HIV-1 replicationduring the acute infection through its effect on reversetranscription. The stimulatory effects of Normosang are medi-ated by a heme/iron-mediated Fenton reaction resulting in theincreased redox stress, while there was no effect on the activa-tion status of the cells (Melkova et al. 2015a; Melkova et al.2015b; Shankaran et al. 2011). Based on our results, wepropose a model in which redox-modulating agents inducechromatin remodeling, affect binding of specific transcriptionfactors to HIV-LTR, and potentiate HIV-1 expression inducedby PKC or other inducers (Melkova et al. 2015a; Melkova et al.2015b).

Historically, there is a case of one HIV+ patient that wasadministered Normosang and consequently remained p24-negative for several months (Pavel Martasek, FacultyGeneral Hospital Prague, personal communication). We sug-gest that the outcome in this particular patient resulted fromthe inhibition of reverse transcription by heme (Levere et al.1991) together with a short-term reactivation and death of theinfected cells due to heme/iron-mediated redox stress.Consequently, a stable heme degradation product, antioxidantbilirubin, might havemediated prolonged inhibitory effects onHIV-1 reactivation (Melkova et al. 2015a; Melkova et al.2015b). This scenario would be compatible with the hypoth-esis of Chirullo et al. (2013) that auranofin decreases numbersof latently infected TCM and TTM CD4+ T cells by its pro-oxidant effects and thus reduces size of the HIV latent pool.

The idea of possible involvement and use of redox stress topurge the latent reservoir is further supported by work of(Iordanskiy et al. 2015) showing that ionizing radiation alonewas sufficient to activate the HIV-1 LTR and to effectively killthe infected T cells. Consequently, this group proposed that alow-dose ionizing radiation could be used therapeutically toreactivate and kill latently infected reservoir cells (Iordanskiyand Kashanchi 2016). Additionally, in the Berlin patient, theonly known case of HIV-1 cure, high-dose irradiationwith bonemarrow transplantation from a donor harboring CCR5Δ32mu-tation with missing CCR5 expression was used, while irradia-tion was omitted in Boston patients in which virus reboundoccurred (Henrich et al. 2013; Hutter et al. 2015).

Finally, any common infection involves increased genera-tion of reactive oxygen and nitrogen species. Co-infections arewell-known to increase HIV-1 replication and promote itsspread (Modjarrad and Vermund 2010), while decreased levelsof GSH, indicator of an increased redox stress, have beendescribed early in HIV infection (Pace and Leaf 1995).Reactive oxygen and nitrogen species were shown to stimulate

activation of the redox-sensitive transcription factor NF-κBand LTR-driven expression of reporter genes (Jimenez et al.2001; Melkova et al. 2000; Pyo et al. 2008). Thus, increasedgeneration of free radicals is likely to be helpful in the attemptsto eliminate the latent HIV-1 in the presence of cART.

A major concern when considering testing of LRA in vivois the induction of immune hyperstimulation upon reactivationof the latent HIV-1 and development of a harmful cytokinestorm. On the other hand, it is not clear if any significantlatency reversal is achievable without increased cytokinelevels (Marsden et al. 2015). However, specific anti-HIV im-mune responses are generally hampered or dysfunctional.Further, it was shown that both HDACi (romidepsin,panobinostat) and PKC agonists (prostratin and bryostatin-1,but not ingenol B) inhibited HIV-specific T cell proliferation(Clutton et al. 2015).

Based on clinical studies, it has been widely accepted thatstimulation or improved function of immune responses in ad-dition to LRAwould be necessary in order to achieve death ofreactivated cells and thus decrease the size of the latent reser-voir. Synergistic effects of different agents should also de-crease the doses and/or negative side effects of individualcompounds.

Summary/conclusions

In conclusion, in view of the currently described sources of theremaining low-level HIV-1 replication in the presence of sup-pressive cART, additional mechanisms and processes promot-ing HIV-1 persistence remain to be identified. Any attempt toachieve HIV-1 cure by latency reversal should involve com-bination of several LRA with different mechanism of actiontogether with stimulation or improvement of immune re-sponses toward infected cells. To validate the efficiency ofindividual approaches, development of sufficiently sensitiveand specific methods allowing accurate determination of thesize of the latent reservoir and its changes is necessary.

Acknowledgments We are grateful to Dr. Ivan Hirsch for helpful dis-cussions and comments. The work of P.S. and M.M. was performed inpartial fulfillment of the requirements for Ph.D. degree at the FirstMedical Faculty, Charles University. The work was supported by theGrant Agency of the Ministry of Health of the Czech Republic – projectNo. NT/14135-3.

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